Studies will be conducted to determine the mechanism of action of chalcone isomerase and the factors which are responsible for catalysis. The isomerization of chalcones to flavanones is an intramolecular reaction and a membrane of the class of biochemical reactions involving addition to a double bond conjugated to a carbonyl groups. Active-site peptides modified by several chemical modification reagents will be isolated, purified and sequenced. Affinity labels will be used to identify a putative enzymic base. The structure-activity relationship in chalcone isomerase will be investigated by a characterization of site-directed mutants. The proposal that the enzyme utilizes covalent catalysis will be tested using a suicide substrate designed to trap the putative covalent enzyme intermediate. The stereochemistry of the addition to the chalcone double bond will be determined and its implications evaluated. The nature of the rate-limiting step will be investigated to determine if the reaction is limited by diffusion, chemistry, or product release. Studies will include the effect of viscosity and deuterium isotopes exchange studies will be performed to determine if the enzyme catalyzes proton exchange from C-3 of the flavanone product. The protonation state and conformation of enzyme-bound inhibitors will be probed by analysis of the spectra of enzyme-bound species. The hypothesis that the enzyme utilizes a "one-base mechanism" will be evaluated using isotope transfer. Chalcone isomerase is involved in the phytoalexin response and may have a role in the expression of disease resistance. Chalcones also have a number of biological activities on mammals: they have an anti-inflammatory activity and are useful in the treatment of allergies and gastrointestinal ulcers. The research outlined in this proposal will undoubtedly contribute much to our knowledge of the chemistry and reactivity of this class of compounds, as well as the mechanism of action of chalcone isomerase.